Project Summary Expression of protein-coding genes is a multi-step process beginning with RNA polymerase II transcription and RNA processing in the nucleus followed by export of the mature mRNA to the cytoplasm for translation. All of the steps in gene expression are coordinated via an extensive network of both physical and functional interactions between the machineries that carry out each step of the pathway. Defects in gene expression are a major cause of human disease, and conversely, gene expression is one of the major cellular processes that can be harnessed to diagnose and treat disease. Thus, a detailed understanding of gene expression is essential for both understanding and treating disease. The long-term objective of the proposed research is to achieve a detailed understanding of the mechanisms for coupling transcription to splicing and for coupling splicing to mRNA export. In addition, the mechanisms and factors required for export of mRNAs derived from genes that naturally lack introns is a central objective. In Specific Aim 1, a coupled RNAP II transcription/splicing system will be established to use in conjunction with RNA interference to identify proteins that function in coupling. The oncoprotein TLS/FUS is a strong candidate protein that will be tested. The system will also be used to test a new model, the U1/SR stamping model, which proposes that the co-transcriptional recruitment of U1 snRNP and SR proteins to the 5[unreadable] splice site functions to achieve the extremely high fidelity required for splicing. An immobilized coupled transcription/splicing assay will also be established in order to investigate the mechanisms that function in co-transcriptional RNA processing. Finally, the recent exciting discovery that transcription elongation factors are specifically associated with U2 snRNP will be investigated to determine whether addition of U2 snRNP to the pre-mRNA during spliceosome assembly is coupled to transcription elongation. In Specific Aim 2, the mechanism for coupling mRNA export to splicing will be investigated by combining biochemical studies with a powerful system for assaying mRNA export in mammalian cells. The function of the highly conserved mRNA export machinery (the TREX complex) will be determined using this system. The role of ATP and the TREX component UAP56, which is a DEAD box helicase/ATPase, in assembly of the TREX complex will also be determined. In addition, the mechanism for specifically recruiting the TREX complex to spliced mRNAs and not to unspliced pre-mRNAs will be elucidated. In Specific Aim 3, the mechanism and factors involved in export of mRNAs derived from genes that naturally lack introns will be determined. At present, little is know about this export pathway. A newly established mammalian system for assaying export of intronless mRNAs will be combined with biochemical studies to define the cis-acting sequences involved in mRNA export and the factors that function in this process. Narrative The steps in gene expression are coupled to one another via an extensive network of physical and functional interactions. The major goal of this project is to achieve a detailed understanding of the mechanisms and factors involved in the functional coupling between transcription, splicing and mRNA export.